Project Summary The long term goal of our research is to understand how MAPK signaling dynamics controls and coordinates cell fate during tissue homeostasis and development. Mitogen Activated Protein Kinases (MAPKs) are clinically relevant signaling molecules that orchestrate cellular responses to a diverse array of stimuli. There are three major MAPK signaling cascades (ERK, p38 and JNK) that control opposing cellular decisions such as survival/apoptosis or proliferation/senescence. Even though these opposed functional roles have been well- characterized, a wide variety of stimuli (i.e. cytokines, cellular stresses or growth factors) have been shown to activate all branches of this highly interconnected network. In addition, whether cells finally apoptose, senesce or enter cell cycle is a highly heterogeneous outcome, even in isogenic cells experiencing the same environment. Our current understanding of how the MAPK network controls cell fate is incomplete because: (i) a lack of integrated methods to quantify the dynamics of the network as a whole and (ii) the use of cell population assays that average unsynchronized single cell behaviors. To address this need, my laboratory has pioneered a new generation of biosensors that allow simultaneous quantification of multiple kinase activities in thousands of live single cells. These biosensors convert phosphorylation into a nucleocytoplasmic shuttling event that can be easily measured by fluorescent microscopy. Our unique methodology features the high temporal resolution, sensor multiplexing capabilities, and single cell resolution essential for studying signaling network dynamics in single cells of a multicellular system. Our central hypothesis is that the signaling equilibrium between MAPKs is critical to regulate single cell outcomes (i.e. proliferation, quiescence, senescence, apoptosis). However, the crosstalk dynamics between individual MAP kinases has not been systematically studied. This is, in part, because MAPK studies use a wide variety of experimental conditions, cell types and genetically altered cells. In this project we will dissect MAPK signaling dynamics at the molecular, cellular and multicellular levels: In Project 1 we will systematically interrogate the crosstalk dynamics between MAP kinases and the phenotypic consequences of this crosstalk. In Project 2 we will use cell culture and primary organoid models to understand the role of MAPK signaling in maintaining tissue homeostasis during early oncogenesis. In Project 3 we will study how MAPK signaling dynamics robustly specifies the mammalian embryo during preimplantation development.